1. Field of the Invention
The invention provides materials and methods relating to identification and optimization of selective inhibitors of glycogen synthase kinase 3 (GSK3), and also relates to methods of treating a condition mediated by GSK3 activity. Such conditions include Alzheimer""s disease, type 2 diabetes, and inflammation.
2. Description of the Related Art
Glycogen synthase kinase 3 (GSK3) is a proline-directed serine/threonine kinase originally identified as an activity that phosphorylates glycogen synthase as described in Woodgett, Trends Biochem Sci 16:177-181 (1991). The role in glucose metabolism has been elaborated recently in Summers et al., J Biol. Chem. 274:17934-17940 (1999). GSK3 consists of two isoforms, xcex1 and xcex2, and is constitutively active in resting cells, inhibiting glycogen synthase by direct phosphorylation. Upon insulin activation, GSK3 is inactivated, thereby allowing the activation of glycogen synthase and possibly other insulin-dependent events. GSK3 is inactivated by other growth factors or hormones that, like insulin, signal through receptor tyrosine kinases. Examples of such signaling molecules include IGF-1 and EGF as described in Saito et al., Biochem. J. 303:27-31 (1994), Welsh et al., Biochem. J. 294:625-629 (1993), and Cross et al., Biochem. J. 303:21-26 (1994). GSK3 has been shown to phosphorylate xcex2-catenin as described in Peifer et al., Develop. Biol. 166:543-56 (1994). Other activities of GSK3 in a biological context include GSK3""s ability to phosphorylate tau protein in vitro as described in Mandelkow and Mandelkow, Trends in Biochem. Sci. 18:480-83 (1993), Mulot et al., Febs Lett 349: 359-64 (1994), and Lovestone et al., Curr. Biol. 4:1077-86 (1995), and in tissue culture cells as described in Latimer et al., Febs Lett 365:42-6 (1995). Selective inhibition of GSK3/may be useful to treat or inhibit disorders mediated by GSK3 activity.
There is a need in the art for compositions and molecules that bind to or interact with GSK3, thereby mediating GSK3 activity. The invention meets this need by providing crystallizable GSK3 polypeptides useful for design and optimization of GSK3 inhibitors.
The invention provides GSK3xcex2 molecules with N- and C-terminal truncations, wherein the molecules are capable of crystallization.
The invention further provides GSK3xcex2 molecules truncated at amino acid R344, R354, T364, A374, and I384.
The invention provides a polypeptide consisting essentially of SEQ ID NO:2 or SEQ ID NO:3, polynucleotides encoding these polypeptides, and vectors comprising these polynucleotides.
The invention still further provides GSK3xcex2 molecules wherein translation of the molecule begins at G34, T39, P44, D49 or V54.
The invention also provides GSK3xcex1 molecules with N- and C-terminal truncations, wherein the molecules are capable of crystallization.
The invention further provides a GSK3xcex1 molecule wherein translation of the molecule begins at S97 and ends at S447, polynucleotides encoding this polypeptide, and vectors comprising these polynucleotides.
The invention further provides a method of identifying a GSK3 polypeptide capable of crystallization, comprising: (a) providing a truncated GSK3 polypeptide; (b) testing the polypeptide for formation of crystals.
The invention also provides GSK3 polypeptides capable of interacting with inhibitors of GSK3.
The invention further provides a method of identifying an enzymatically active GSK3 polypeptide, comprising: (a) providing a truncated GSK3 polypeptide; (b) contacting the polypeptide with a substrate of GSK3; and (c) measuring the kinase activity of the polypeptide after contacting the polypeptide with the substrate, wherein the polypeptide is active if it shows  greater than 0.01xc3x97the activity of the full-length enzyme and preferably  greater than 0.1xc3x97the activity of the full-length enzyme.